1. Field of the Invention
The present invention relates to a process for producing a recombinant fibrinogen producing cell producing a large amount of fibrinogen which is one of plasma proteins. More particularly, the present invention relates to a process for producing a recombinant fibrinogen producing cell comprising a step of incorporating, into an animal cell, genes encoding three kinds of proteins constituting fibrinogen, an α chain (or variant of α chain), a β chain and a γ chain (or variant of γ chain) at a constitutional ratio thereof so that the number of a γ chain (and/or variant of γ chain) gene is an equivalent amount to a 1000-fold amount relative to a total number of an α chain (and/or variant of α chain) gene and a β chain gene, and a step of incorporating a production amount enhancing factor, and a recombinant fibrinogen highly producing cell obtained by the process, and fibrinogen obtained therefrom.
2. Description of the Related Art
Fibrinogen is responsible for serving to coagulate a blood when a living body undergoes injury, as one of blood coagulation factors. The first function is to form a body of a thrombus called fibrin clot at a site of injury, and the second function is to serve as an adhesive protein necessary for platelet aggregation. A blood concentration of fibrinogen is usually about 3 mg/ml, and is third highest next to albumin, and immunoglobulin G.
Fibrinogen is a macro-glycoprotein consisting of a total 6 of polypeptides having each two of three kinds of different polypeptides called α chain, β chain and γ chain. An individual molecular weight of polypeptides is about 67000 in the case of an α chain, about 56000 in the case of a β chain, and about 47500 in the case of a γ chain, and a molecular weight of fibrinogen as an aggregate of them reaches about 340000 (see “Hemostasis•Thrombus•Fibrinolysis”, Matsuda and Suzuki ed., Chugaiiyakusha (1994)). In a fibrinogen molecule, a half molecules of an α chain, a β chain and a α chain which are S—S bonded (α-β-γ) form a further S—S bonded dimer (α-β-γ)2, and the fibrinogen molecule has a shape of three nodular (knotting spherical) structures. That is, the fibrinogen molecule takes a structure consisting of a central E region, two D regions symmetrically disposed on both outer sides thereof, and a rod-like part connecting therebetween.
In fibrinogen in blood, heterogenous molecules due to possession of variant polypeptides having different molecular sizes are present. For example, in a γ chain, the presence of a variant called γ′ chain (or γB chain) is reported, and this has been revealed to be a polypeptide comprising a total 427 of amino acid residues in which 20 amino acid residues are added to a 408-position of an amino acid sequence of a γ chain (see Chung D E and Davie E W, Biochemistry, 23, 4232 (1984)). In addition, in an α chain, a variant called αE is present, and it is reported that this polypeptide has a total 847 of amino acid residues in which 236 amino acids are extended at a 612-position of an amino acid sequence of an α chain (see Lawrence Y F et al., Biochemistry, 31, 11968 (1992)). A remarkable difference in its aggregating ability and the fibrinolysis resistance ability is not recognized in heterogenous fibrinogen having γ′ and αE as compared with normal fibrinogen. However, these molecular species are under study, and the detailed function has not been elucidated yet.
A fibrinogen preparation is effective in arresting serious bleeding by enhancing a fibrinogen concentration in blood by a method such as intravenous administration, and is used in improving the consumption state of a blood coagulation factor such as disseminated intravascular coagulation (DIC) in sepsis, or supplemental therapy in congenital or acquired fibrinogen deficiency. In addition, a fibrinogen preparation is widely utilized as a tissue adhesive utilizing agglutination property of fibrin (see “Special Edition•Bioadhesive” Biomedical Perspectives, 6, 9-72 (1997)). This living body-derived adhesive utilizes gelling of fibrinogen in a living body, and is widely used in hemostasis, closure of a wound site, adhesion or suturing reinforcement of nerve, tendon, vessel and tissue, and closure of air leakage in lung. In addition, in recent years, a preparation which has enhanced convenience by adhering fibrinogen to a sheet of collagen has been sold.
Currently, fibrinogen used as a medicament is prepared from human plasma, and a problem has been exemplified that 1) since plasma collected from unspecific many humans is used, there is a crisis that infective pathogen such as a virus causing pneumonia such as HAV, HBV, HCV, HEV and TTV, a virus causing immunodeficiency such as HIV, and abnormal prion causing CJD is mixed in, and 2) in Japan, plasma is supplied by blood donation, and future stable supply is problematic.
In order to solve these problems, recombination of fibrinogen has been previously tried. For example, in Escherichia coli, expression of a fibrinogen γ chain in a bacterial cell has been succeeded, but there is no report that three proteins of an a chain, a β chain and a γ chain are simultaneously expressed to produce a functional fibrinogen molecule. In addition, also in an expression system using yeast, there was transiently a report that secretion and expression have been successful, but finally reproductivity was not confirmed, and the report was canceled (see Redman C M, Kudryk B., J. Biol. Chem., 274, 554 (1999)). Like this, there has been not a report yet that fibrinogen has been successfully expressed using Escherichia coli or yeast.
On the other hand, in an animal cell, expression has been tried using a BHK cell (see Farrell D H et al., Biochemistry, 30, 9414 (1991)), a COS cell (see Roy S N et al., J. Biol. Chem., 266, 4758 (1991)) or a CHO cell (see
Lord S T et al., Blood Coagul Fibrinolysis, 4, 55 (1993), Binnie C G et al., Biochemistry, 32, 107 (1993) and Lord S T et al., Biochemistry, 35, 35, 2342 (1996), and U.S. Pat. No. 6,037,457), but the production amount is only around 1 to 15 μg/ml. In these cases, using any of a metallothionein promoter, a Rous sarcoma virus LTR promoter, and an adenovirus 2 major late promoter, and any of an aminoglycoside 3′ phosphotransferase (neo) gene, a dihydrofolate reductase (dhfr) gene, and a histidinol resistance gene or a combination thereof is used as a selectable marker. In any case, a method of independently constructing expression vectors for genes encoding an α chain, a β chain and a γ chain, respectively, and transfecting a cell with three vectors simultaneously, or introducing an expression vector having a β chain gene and an α chain gene later into a cell which has been previously transformed with two expression vectors, each having an α chain gene and aβ chain gene, or a β chain gene and a γ chain gene, and a method of mixing a plasmid having an α chain gene and a β chain gene and a plasmid having a β chain gene at an equal amount, and introducing the mixture is adopted. In any case, there is, particularly, no description regarding a constitutional ratio of respective genes upon introduction, and it is thought that respective genes are equally introduced according to a general procedure. In a medicament using fibrinogen derived from blood which is currently used, for example, in a fibrin paste preparation, about 80 mg/dose of fibrinogen is used and, at an expression amount of the aforementioned more than a dozen μg/ml, a production facility must be scaled up, necessarily leading to the high cost. In order to produce fibrinogen at a practical level by the gene recombinant technique, a highly producing cell (e.g. expression amount of fibrinogen is 100 μg/ml or more) is necessary, but currently, there is no report of an expression system using a recombinant animal cell satisfying this.
On the other hand, when a recombinant fibrinogen producing cell is cultured, the same problem as that of culturing of a normal animal cell is contemplated. In general, when a protein is a secreted protein, since an objective protein can be recovered in the culture supernatant, a method of culturing a recombinant animal cell in a suitable medium, culturing it for a certain term and, thereafter, recovering the culture supernatant at once (batch culturing), or extracting a suitable amount of a medium at an arbitral time, and performing addition continuously (perfusion culturing) is used. In any event, as the number of a recombinant animal cell producing an objective secreted protein is increased, an amount of a secreted protein accumulated (produced) in a medium is increased. Growth of a cell is divided into three phases: a logarithmic phase when a cell is grown logarithmically, a stationary phase when the number of cells is apparently constant, and a death phase when a cell dies, and the number of cells is decreased. In order to increase production of a secreted protein, it is important to enhance a cell density of a recombinant animal cell at a stationary phase as high as possible, and maintain the term as long as possible. Particularly, in the case of batch culturing, since a recombinant animal cell is grown in a constant amount of a medium, many attempts have been tried to enhance a cell density at a stationary phase as high as possible in order to increase a production amount of a secreted protein therein, and maintain the term as long as possible.
As a method different from such the rearing method, an attempt has been also tried to modify a host cell. For example, a method using an anti-apoptotic factor has been tried. This method tries to express an anti-apoptotic factor gene in a recombinant animal cell producing a protein, and imparting the ability of suppressing programmed cell death (apoptosis) generated by nutrient starving to the cell to extent a stationary phase.
A mechanism causing apoptosis is thought as follows according to “Apoptosis and Disease Central Nervous System Disease” Yoshikuni Mizuno ed., Drug Journal (2000). When a variety of cell death stimulations such as nutrient depletion is transmitted to a cell, the signal is transmitted to mitochondria via various proteins including a transcription factor and a kinase. Mitochondria which has received the signal releases an apoptosis signal transmitting factor (AIF, cytochrome c etc.) into a cytoplasm. Cytochrome c together with Apaf-1 (apoptosis activating factor-1) present in a cytoplasm is bound to pro-caspase-9 to form a complex, activating caspase-9. An activated caspase cascade cuts various substrates in a cytoplasm or a nucleus, and induces a morphological or a biochemical change (actin degradation, DNA fragmentation, chromosome aggregation etc.) characteristic in various apoptoses. As a factor which suppresses such the apoptosis, Bcl-2 (B cell lymphoma/leukemia 2) is well-known. A Bcl-2 gene was found as an oncogene which is frequently seen in human follicular lymphoma. Currently, many family genes having a domain (BH1-4) having high homology with Bcl-2 have been identified. In a family, there are factors which suppressively serve in apoptosis, and factors which promotively serve and, as an suppressible factor, for example, Bcl-xL, Bcl-w, Mcl-1, Al, BHRF1, E1B-19K, and Ced-9 are known, and it is though that the factor arrests signal transmission by inhibiting the aforementioned release of cytochrome c or binding with Apaf-1 and procaspase-9. It is thought that such the suppressible Bcl-2 family functions upstream of a caspase cascade.
On the other hand, a factor which acts downstream of a caspase cascade (inhibits activity of caspase directly) to show anti-apoptotic effect is also known. For example, a P35 protein of AcNPV (Autogropha californica nuclear polyhedrosis virus) belonging to a Baculovirus family is cut as a substrate of caspase, a fragment thereof forms a stable composite with almost all caspases to inhibit its activity. Therefore, various apoptosis can be suppressed. BmNPV (Bombyx mori nuclear polyhedrosis virus) which is close to AcNPV also has a P35 gene. In addition, crmA of cowpox virus specifically binds to caspase-1-like protease and caspase-8, -10, and inhibits this, thereby, apoptosis can be suppressed. In addition, v-FLIP derived from herpesvirus has two DEDs (death effector domains), and suppresses activation of caspase-8 by binding to FADD (Fas-associating Protein with death domain).
Further, in many close viruses including CpGV (Cidia pomonella granulosis virus) and OpMNPV (Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus) of Baculovirus family, a v-IAP (inhibitor of apoptosis) gene whose expression product inhibits directly caspase activity has been identified besides a P35 gene. Up to now, as a homologue of v-IPA, an IPA family having a few kinds of BIRs (baculovirus IPA repeats) such as c-IAP1/hia-2, c-IAP2/hia-1, XIAP, NAIP, survivin, TIAP, Apollon, DIAP1, DIAP2, SfIAP, and ITA has been identified in a drosophila and a mammal in addition to a virus.
However, methods of potentiating a production amount using an anti-apoptotic factor derived from a Bcl-2 family such as Bcl-2, Bcl-xL, and E1B-19K which acts on upstream of a cascade are all inhibit cell death, and can extend a stationary phase of a growth carve, but a production amount is not increased as expected, in many cases. From these things, it is thought that these factors have no direct effect of potentiating production amount of a protein, or if present, exerts the effect under the special environment. On the other hand, regarding a caspase inhibiting action factor which acts on downstream of a cascade, there is few reports in which a relationship with the production amount potentiating effect was investigated in a recombinant protein producing cell, and the effect has been unknown.